Neovascularization inhibitor

ABSTRACT

The invention relates to a neovascularization inhibitor composition comprising a compound of the following general formula  I! or a salt thereof, or a solvate thereof, as an active ingredient, ##STR1## wherein R 1  represents hydrogen, optionally substituted alkyl, aralkyl, arylalkenyl, or aryl; R 2  represents optionally substituted alkyl, aralkyl, arylalkenyl, or aryl; or R 1  and R 2  conjoinedly and taken together with the adjacent N atom, i.e. in the form of NR 1  R 2 , represent a 4- through 8-membered cyclic amino group optionally containing nitrogen, oxygen, or sulfur as a ring member in addition to said N atom and optionally being further substituted.

This application is a 371 of PCT/JP96/01069 filed Apr. 19, 1996.

TECHNICAL FIELD

The present invention relates to a neovascularization inhibitorcomposition.

BACKGROUND ART

The blood vessels, together with the heart and the lymph vessels,constitute a vasculature which is indispensable for sustained metabolismof tissues and, hence, functional homeostasis of an organism. The chiefconstituent cells of a blood vessel are endothelial cells and smoothmuscle cells. Proliferation of endothelial cells in the establishedvascular system is usually observed in the neovascularization processgiving rise to a new network of capillary blood vessels chiefly fromvenules and the repair process following exfoliation of vascularendothelial cells. As it has recently been made clear thatneovascularization is closely involved in pathology of growth of solidtumors, arteriosclerosis, hyperplasia of panni in rheumatoid arthritis,ophthalmic diseases such as diabetic proliferative retinopathy,psoriasis vulgaris, etc., there is a mounting interest inneovascularization.

The mechanism of neovascularization in cancers, rheumatoid arthritis,diabetic retinopathy, etc. is known to begin with a disruption ofextracellular matrix which triggers migration of endothelial cells toform a tube which, in turn, is followed by the migration andproliferation of vascular smooth muscle cells around the tube tocomplete a new blood vessel.

Several compounds (e.g. D-penicillamine, heparin, 15-deoxyspergaulin,eponemycin AGM-1470, tecogalan sodium (DS-4152), herbimycin A, andinterferon-alpha) are known to inhibit neovascularization. Thosecompounds are either biological component-related substances orsubstances of the natural origin and, therefore, the source of theirsupply is too limited for practical utilization.

The antitumor agent irsogladine maleate2,4-diamino-6-(2,5-dichlorophenyl)-1,3,5-triazine maleate!, which isstructurally close to the compound of the invention, reportedly inhibitsneovascularization (FEBS, 322(2), 155-158, 1993). However, the2-amino-4-substituted amino-6-(2,5-dichlorophenyl)-1,3,5-triazinederivative which is theoretically available upon substitution of one ofthe amino groups by alkyl, aralkyl, arylalkenyl, or aryl is not known tohave neovascularization inhibitory activity.

Meanwhile, the inventors of the present invention previously discoveredthat the 2-amino-4-substitutedamino-6-(2,5-dichlorophenyl)-1,3,5-triazine derivative has antihepatitisactivity and is useful as a therapeutic agent for hepatitis and alreadyfiled a patent application (WO 96/04914).

DISCLOSURE OF INVENTION

The object of the present invention is to provide an excellent medicinewith low toxicity which is effective in inhibiting neovascularization.

The inventors of the present invention found that the compound of thefollowing formula I! has remarkably high neovascularization inhibitoryactivity with extremely low toxicity and have completed the presentinvention. ##STR2## wherein R¹ represents hydrogen, optionallysubstituted alkyl, aralkyl, arylalkenyl, or aryl; R² representsoptionally substituted alkyl, aralkyl, arylalkenyl, or aryl; or R¹ andR² conjoinedly and taken together with the adjacent N atom, i.e. in theform of NR¹ R², represent a 4- through 8-membered cyclic amino groupoptionally containing nitrogen, oxygen, or sulfur as a ring member inaddition to said N atom and being further substituted.

The present invention, therefore, is directed to a neovascularizationinhibitor composition comprising a compound of the above formula I! or asalt thereof, or a solvate thereof, as an active ingredient.

The present invention is predicated on the finding that the2-amino-4-substituted amino-6-(2,5-dichlorophenyl-1,3,5-triazinederivative, which has antihepatitis activity as mentioned above,additionally has neovascularization inhibitory activity which is quiteunrelated to the first-mentioned activity.

As demonstrated in the experimental examples presented hereinafter, thecompound of the invention has by far higher neovascularizationinhibitory activity than irsogladine maleate.

The present invention is now described in further detail.

The "alkyl" mentioned for R¹ and R² includes straight-chain or branchedalkyl groups of 1-10 carbon atoms, such as methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl,isopentyl, n-hexyl, isohexyl, n-heptyl, isoheptyl, n-octyl, isooctyl,n-nonyl, isononyl, n-decyl, and isodecyl. Preferred are C₁₋₄ alkylgroups. Such alkyl groups may each be substituted by one, two or threesubstituents, whether similar or dissimilar, as selected from the groupconsisting of hydroxy, alkoxy, amino, monoalkylamino, dialkylamino,arylamino, cyclic amino, carboxy, carbamoyl, aryloxy, and acyloxy. Thealkoxy mentioned above includes straight-chain and branched alkoxygroups of 1 to 4 carbon atoms, such as methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, isobutoxy, sec-butoxy, and tert-butoxy. The alkylof said monoalkyl- or dialkylamino includes straight-chain and branchedalkyl groups of 1-4 carbon atoms, such as methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. The aryl ofsaid arylamino or aryloxy includes aryl groups of 6-13 carbon atoms,such as phenyl, 1-naphthyl, 2-naphthyl, and biphenyl. Particularlypreferred is phenyl. Those aryl groups may each be substituted by one or2-3 similar or dissimilar substituents selected from the groupconsisting of straight-chain or branched C₁₋₄ alkyl (e.g. methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl) and C₁₋₄alkoxy (e.g. methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy,isobutoxy, sec-butoxy, tert-butoxy). The cyclic amino as a substituentfor said alkyl includes saturated or unsaturated groups such asazetidin-1-yl, pyrrolidin-1-yl, 3-pyrrolin-1-yl, piperidino,hexamethyleneimino, octahydroazocin-1-yl, piperazin-1-yl,homopiperazin-1-yl, morpholino, and thiomorpholino. Particularlypreferred are piperidino, piperazin-1-yl, and morpholino. This cyclicamino group may be substituted by C₇₋₁₄ aralkyl such as benzyl,phenethyl, phenylpropyl, phenylbutyl, 1-naphthylmethyl,2-naphthylmethyl, and diphenylmethyl. The particularly preferredsubstituent is benzyl. The acyl of said acyloxy includes C₁₋₆ alkanoyl(e.g. formyl, acetyl, propionyl, butyryl, valeryl, hexanoyl), C₇₋₁₀aroyl (e.g. benzoyl), and heterocyclylcarbonyl (e.g. nicotinoyl), amongothers. Particularly preferred is benzoyl.

The "aralkyl" for R¹, R² includes C₇₋₁₄ aralkyl such as benzyl,phenethyl, phenylpropyl, phenylbutyl, 1-naphthylmethyl,2-naphthylmethyl, or diphenylmethyl. Particularly preferred is benzyl.

The "arylalkenyl" for R¹, R² includes C₈₋₁₀ arylalkenyl such as styryl,cinnamyl, or 4-phenyl-2-butenyl.

The "aryl" for R¹, R² includes C₆₋₁₃ aryl such as phenyl, 1-naphthyl,2-naphthyl, or biphenyl. Particularly preferred is phenyl.

The "cyclic amino" for NR¹ R² includes saturated or unsaturated cyclicamino groups. Thus, there may be mentioned azetidin-1-yl,pyrrolidin-1-yl, 3-pyrrolin-1-yl, piperidino, hexamethyleneimino,octahydroazocin-1-yl, piperazin-1-yl, homopiperazin-1-yl, morpholino,thiomorpholino, etc. This cyclic amino may be substituted by one or 2-4similar or dissimilar substituents selected from the group consisting ofhydroxy, oxo, carboxy, alkyl, hydroxyalkyl, aryloxyalkyl, aminoalkyl,alkylsulfonylaminoalkyl, alkylsulfonylamino, alkylsulfonylaralkyl,alkylsulfonyl, aryl, aralkyl, and 2-pyrimidinyl. The alkyl moiety of anysuch substituent includes straight-chain or branched C₁₋₄ alkyl such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, ortert-butyl. The aryl of said substituent groups for said cyclic aminoincludes C₆₋₁₂, aryl groups such as phenyl, 1-naphthyl, 2-naphthyl, andbiphenyl. This aryl may be substituted by one or 2-3 similar ordissimilar substituents selected from the group consisting ofstraight-chain or branched C₁₋₄ alkyl (e.g. methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl) and C₁₋₄ alkoxy(e.g. methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy,sec-butoxy, tert-butoxy). The aralkyl includes C₇₋₁₄ aralkyl groups suchas benzyl, phenethyl, phenylpropyl, phenylbutyl, 1-naphthylmethyl,2-naphthylmethyl, and diphenylmethyl. The aryl moiety of this aralkylmay be substituted by one or 2-3 similar or dissimilar substituentsselected from the group consisting of straight-chain or branched C₁₋₄alkyl (e.g. methyl, ethyl, n-propyl, isopropyl n-butyl, isobutyl,sec-butyl, tert-butyl) and C₁₋₄ alkoxy (e.g. methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy).

Preferably, R¹ and R² each represents C₁₋₄ alkyl substituted by onesubstituent. Particularly preferred is alkyl substituted by hydroxy.Most preferable is hydroxyethyl.

NR¹ R² is preferably a 5- or 6-membered cyclic amino group which iseither unsubstituted or substituted by one or 2 similar or dissimilarsubstituents and more preferably represents pyrrolidin-1-yl, piperidino,or morpholino. Particularly preferred is pyrrolidin-1-yl. The preferredsubstituent for this cyclic amino is hydroxy or hydroxyalkyl. Thehydroxyalkyl is preferably hydroxymethyl.

The compound I! of the invention may be used in the free form or in theform of a pharmacologically acceptable acid addition salt, such as saltswith inorganic acids (e.g. hydrochloride, sulfate, nitrate, phosphate,hydrofluoride, hydrobromide) or salts with organic acids (e.g. acetate,tartrate, lactate, citrate, fumarate, maleate, succinate,methanesulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate,naphthalenesulfonate, camphorsulfonate). The compound (I) can also beused in the form of a solvate.

The solvate of the compound of the invention includes the hydrate,ethanolate, and other pharmacologically acceptable solvates.

The compound I! of the invention can be prepared, for example by thefollowing process WO96/04914!. ##STR3##

In the above reaction scheme, R¹ and R² are as defined hereinbefore; Xrepresents halogen such as chlorine or bromine.

Thus, I! can be prepared by reacting a compound II! with amine III! inthe presence of a base in an inert solvent at 0°-200° C., preferably25°-100° C. The reaction solvent that can be used includes aprotic polarsolvents such as acetonitrile, dimethyl sulfoxide andN,N-dimethylformamide (DMF), ethers such as tetrahydrofuran,dimethoxyethane, diethyl ether and dioxane, glymes such asmethylcellosolve and ethylcellosolve, halogenated hydrocarbons such asmethylene chloride and chloroform, hydrocarbons such as benzene, tolueneand xylene, and mixtures of those solvents. The base that can be usedincludes inorganic bases such as alkali metal carbonates (e.g. potassiumcarbonate, sodium carbonate), alkali metal hydrogen carbonates (e.g.potassium hydrogen carbonate, sodium hydrogen carbonate) and alkalimetal hydroxides (e.g. potassium hydroxide, sodium hydroxide) andorganic bases such as triethylamine and pyridine. In lieu of such abase, the amine (HNR¹ R²) may be used in excess.

The reaction time depends on species of the starting compounds, base,and solvent but may range from several minutes to 24 hours.

The molar ratio of amine III! to compound II! is generally at leastequimolar and preferably 1-1.2 molar. The amount of the base isgenerally at least equimolar and preferably 1-2 molar equivalents inproportion to II!.

The compound Ia! of the invention wherein R¹ and/or R² is alkylsubstituted by amino or hydroxy in any desired position or the grouprepresented by the formula NR¹ R² is substituted by aminoalkyl,hydroxyalkyl or hydroxy in any desired position or positions can beprepared by the following alternative process. ##STR4## In the abovereaction schema, X is as defined hereinbefore. R¹¹ and R²¹ eachrepresents an alkyl group substituted by carbamoyl, cyano,alkoxycarbonyl, or oxo in any substitutable position or NR¹¹ RR²¹represents a cyclic amino group substituted by carbamoyl, cyano,alkoxycarbonyl, or oxo in any substitutable position. R¹² and/or R²²represents an alkyl group substituted by amino or hydroxy in theposition corresponding to said carbamoyl, cyano, alkoxycarbonyl or oxoin R¹¹, R²¹, or NR¹² R²² represents a cyclic amino group substituted byaminoalkyl, hydroxyalkyl, or hydroxy in the position corresponding tosaid carbamoyl, cyano, alkoxycarbonyl, or oxo in NR¹¹ R²¹.

The corresponding amine IIIa! having carbamoyl, cyano, alkoxycarbonyl,or oxo in an optional position is reacted with compound II! in the samemanner as above to give compound IV! which is then reduced to providecompound Ia!.

This reduction reaction can be carried out by a per se known method, forexample using a metal hydrogen complex compound such as lithium aluminumhydride or sodium borohydride. The compound Ia! of the invention can beprepared typically by dissolving compound IV! in 2-100 volumes of anether solvent, e.g. diethyl ether, tetrahydrofuran, dimethoxyethane ordioxane, and reacting it with 0.25-1.0 mole of lithium aluminum hydrideat -50° C. to 30° C. for 0.5-10 hours. When sodium borohydride isemployed, the reaction can be carried out in a protoic solvent such asmethanol, ethanol or isopropyl alcohol in lieu of said ether solvent inotherwise the same manner as above.

The starting compound II! can be prepared by the known productiontechnology (JP Kokai S51-70781). Compounds III! and IIIa! can bepurchased from commercial sources or synthesized starting withcommercial compounds.

Some species of the compound of the invention have a asymmetriccarbon(s) and may therefore be optically active but such optical isomersand mixtures thereof also fall within the scope of the invention.

Such optically active compounds can be obtained by isolation with achiral column or by an optical resolution method utilizing theirbasicity which comprises using an optically active acid (tartaric acid,dibenzoyltartaric acid, mandelic acid, 10-camphorsulfonic acid, etc.).They can also be synthesized starting with optically active compoundsIII! or IIIa! prepared in advance.

The compound I! of the invention can be changed to salts in the per seknown manner. For example, the hydrochloride of compound I! according tothe invention can be provided by dissolving compound I! in an alcoholicsolution of hydrogen chloride.

Among species of compound I! of the invention, the carboxy-containingcompounds can be changed to salts in the per se known manner. The saltsmay for example be alkali metal salts such as the corresponding sodiumsalts and potassium salts, and alkaline earth metal salts such as thecorresponding calcium salts. For example, the alkali metal salt ofcompound I! according to the invention can be obtained by addingpreferably one equivalent of sodium hydroxide, potassium hydroxide orthe like to carboxy-containing compound I! in an alcoholic solvent. Thealkaline earth metal salt of compound I! according to the invention canbe provided by dissolving the alkali metal salt obtained as above inwater, methanol, ethanol, or a mixture thereof and adding one equivalentof calcium chloride or the like.

The solvate (e.g. hydrate, ethanolate) of the compound I! or salt of theinvention also falls within the scope of the invention. The solvate maybe obtained, depending on species of compound, by recrystallizing thecompound or salt from the corresponding solvent or a suitable mixedsolvent containing the corresponding solvent. For example, the hydratemay be obtained by recrystallizing compound I! of the invention from anaqueous alcohol.

The compound I! of the invention may assume polymorphism. Suchpolymorphs also fall within the scope of the invention.

The compound I! of the invention, thus prepared, can be isolated andpurified, as a free base or an acid addition salt, by per se knownprocedures such as concentration, pH adjustment, redistribution, solventextraction, crystallization, fractional distillation, andchromatography.

The dosage of the compound in its application as a neovascularizationinhibitor, a therapeutic drug for arteriosclerosis, an antitumor agent,a therapeutic drug for Kaposi's sarcoma, a therapeutic drug for diabeticretinopathy, or a therapeutic drug for rheumatoid arthritis ispreferably selected with reference to patient factors such as age andbody weight, route of administration, nature and severity of disease,and other clinical circumstances. Usually, however, the oral dosage foradult humans, for instance, may range from 0.1 mg to 1 g/patient andpreferably from 1 mg to 100 mg/patient as the active ingredient. Thereare cases in which a higher dosage is needed or a lower dosage issufficient. The above daily dosage is preferably administered in 2 to 3divided doses.

The compound of the invention can be administered either as it is or inthe form of a pharmaceutical composition containing 0.1%-99.5%,preferably 0.5%-90%, of the compound in a pharmaceutically acceptablenontoxic carrier or vehicle to mammalian animals inclusive of man.

The carrier or vehicle that can be used includes one or more solid,semisolid, or liquid diluents, fillers, or other formulationauxiliaries. The pharmaceutical composition is preferably administeredin a unit dosage form. The pharmaceutical composition of the inventioncan be administered orally, parenterally, locally (e.g. transdermaldelivery), or rectally. Of course, the dosage form suited for a selectedroute of administration should be employed. Oral administration, inparticular, is preferred.

Oral administration can be carried out using a solid or liquid unitdosage form such as bulc powders, powders, tablets, dragees, capsules,granules, suspension, solution, syrup, drops or sublingual tablets.

Bulc powders are prepared by comminuting the compound of the inventionto a suitable particle size. Powders can be manufactured by blending theso-comminuted compound of the invention with a similarly comminutedpharmaceutical carrier such as, an edible carbohydrate, e.g. starch,mannitol. Where necessary, a flavorant, preservative, dispersant, color,perfume, etc. may be added.

Capsules can be manufactured by filling capsule shells, e.g. gelatincapsule shells, with the above-mentioned bulc powders or powders or thegranules prepared as described hereinafter for tablets. Prior tofilling, said powders or granules may be formulated with a lubricant orfluidizing agent such as colloidal silica, talc, magnesium stearate,calcium stearate or solid polyethylene glycol. The availability of thedrug after ingestion of capsules can be improved by addition of adisintegrator or solubilizer, such as carboxymethylcellulose,carboxymethylcellulose calcium, low-substitution-degreehydroxypropylcellulose, croscarmellose sodium, carboxymethylstarchsodium, calcium carbonate or sodium carbonate.

The fine powders of the compound of the invention may be suspended ordispersed in a vegetable oil, polyethylene glycol, glycerin, or asurfactant and wrapped in gelatin sheets to provide soft capsules.Tablets can be manufactured by preparing a powdery mixture containing anexcipient, granulating or slugging the mixture, adding a disintegratoror a lubricant, and compressing the whole mixture into the tabular form.The powdery mixture can be prepared by mixing the appropriatelycomminuted compound with the above-mentioned diluent or base, optionallywith addition of a binder (e.g. carboxymethylcellulose sodium,methylcellulose, hydroxypropylmethylcellulose, gelatin,polyvinylpyrrolidone, polyvinyl alcohol), a dissolution retardant (e.g.paraffin), a reabsorption promoter (e.g. quaternary salts), and anadsorbent (e.g. bentonite, kaolin, dicalcium phosphate, etc.). Thepowdery mixture can be first wetted with a binder such as a syrup, astarch paste, gum arabic, a cellulose solution, or a polymer solution,stirred to mix, dried, and crushed to give granules. Instead ofgranulating the powders in this way, it is possible to compress thepowders with a tablet machine in the first place and then pulverize theresulting crude-form slugs to provide granules. The granules can beprotected from interadhesion by adding a lubricant such as stearic acid,its salt, talc or mineral oil. The lubricated mixture is thencompressed. The uncoated tablets thus obtained can be film-coated orsugar-coated.

The compound of the invention can be mixed with a free-flowing inertcarrier and directly compressed without resort to the above-mentionedgranulation or slugging procedure. Transparent or translucent protectivecoats such as a hermetic shellac coat as well as sugar or polymer coatsand wax glaze coats can also be applied. Other oral compositions, suchas a solution, syrup and elixir can also be prepared in unit dosageforms each containing a predetermined amount of the active ingredient. Asyrup is prepared by dissolving the compound of the invention in asuitable flavored aqueous medium, and the elixir can be manufacturedusing a nontoxic alcoholic vehicle. Suspensions can be manufactured bydispersing the compound of the invention in a nontoxic vehicle. Wherenecessary, a solubilizer or emulsifier (e.g. ethoxylated isostearylalcohol, polyoxyethylene sorbitol esters), a preservative, a corrigentor flavor (e.g. peppermint oil, saccharin) and others can also be added.

Where necessary, the unit dosage for oral administration can be providedin a microencapsulated form. This kind of preparation may be coated orembedded in a polymer matrix or a wax to insure a prolonged action orsustained release.

For parenteral administration, a liquid unit dosage form forsubcutaneous, intramuscular, or intravenous administration, in the formof a solution or a suspension, can be employed. Such unit dosage formscan be provided by suspending or dissolving a predetermined amount ofthe compound of the invention in a nontoxic liquid vehicle suitable forinjection, such as an aqueous or oily medium, and sterilizing thesuspension or solution. For isotonizing such injectable preparations, anontoxic salt or a solution thereof can be added. In addition, astabilizer, a preservative, and/or an emulsifier can also beconcomitantly used.

Rectal administration can be made by using suppositories manufactured bydissolving or suspending the compound of the invention in a low-meltingwater-soluble or -insoluble solid base such as polyethylene glycol,cacao butter, semisynthetic fat (e.g. Witepsol™), higher esters (e.g.myristyl palmitate), or a mixture thereof.

The toxicity of the compound of the invention is very low as will bedescribed hereinafter.

The neovascularization inhibitory action of the compound of theinvention could be confirmed by the in vitro tube formation test asdescribed below in Experimental Example 1.

As test compounds, the following compounds were used.

 2-Amino-4-N,N-bis(2-hydroxyethyl)amino!-6-(2,5-dichlorophenyl)-1,3,5-triazine(compound 1)

2-Amino-4-(2,5-dichlorophenyl)-6-(3-hydroxymethyl-1-pyrrolidinyl)-1,3,5-triazinehydrochloride (compound 2)

 2-Amino-4-(2,5-dichlorophenyl)-6-(2S,4R)-2-hydroxymethyl-4-hydroxy-1-pyrrolidinyl!-1,3,5-triazine(compound 3)

EXPERIMENTAL EXAMPLE 1 Tube Formation Test

Phenol red-free Medium-199 supplemented with 10% fetal calf serum (FCS)and ECV-304 cells were used as the culture medium and the cells,respectively. EHS sarcoma extract (Matrigel™, Becton Dickinson (MA)) wasused as the matrix gel.

The wells in the two middle rows of a 24-well multiplate (Corning258201) were filled with 300 μl aliquots of Matrigel, and the gelationwas effected under spinning with a centrifugal machine (2,000 rpm, 30min., 37° C.) (culture by the thick gel method). The culture medium wasadded to the gel, 1 ml per well, and after 24 hours the medium wasrenewed and 10³ cells/well were seeded. After a further 24 hours, thetest drug (1/1,000 in dimethyl sulfoxide) was added. After 1 week ofincubation, ethanol fixation and nuclear staining with ethidium bromidewere carried out and observation was made under the fluorescentmicroscope. As the test drugs, compounds 1, 2, and 3 and the referencecompound irsogladine maleate were used.

The ECV-304 cells in the untreated control group migrated andproliferated radially (in a star-like fashion) to form a stick-liketube, which then branched to form circles in due course, and as thecircles were conjoined to each other, a giant meshwork was formed. Usingthe colonial morphology on day 2 after commencement of culture as anindicator, the effect of the test drug on tube formation was evaluated.As a result, compound 1 was found to completely inhibit branching at10⁻⁵ M, compound 2 did so at 10⁻⁶ M, and compound 3 completely inhibitedformation of circles at 10⁻⁷ and 10⁻⁶ M. On the other hand, the positivecontrol compound inhibited the formation of circles only at 10⁻⁵ M.Those results indicate that the compound of the invention hasconsiderably higher neovascularization inhibitory activity than thereference compound. Moreover, the colony size at this stage wasdefinitely larger in the untreated group than in the treated groups.

The influence of test compounds on colonial morphology on the gel isshown in Table 1.

                  TABLE 1    ______________________________________    The influence of test compounds on colonial morphology    on the gel                       Colonial morphology    Treatment          Number of circles    ______________________________________    Untreated control group                           1    Test drug groups    Compound 1     10.sup.-4 M                           0                   10.sup.-5 M                           0    Reference compound                   10.sup.-5 M                           0    Untreated control group                           5    Test drug groups    Compound 2     10.sup.-6 M                           1                   10.sup.-7 M                           3                   10.sup.-8 M                           6                   10.sup.-9 M                           7    Compound 3     10.sup.-6 M                           0                   10.sup.-7 M                           1                   10.sup.-8 M                           2                   10.sup.-9 M                           6    Reference compound                   10.sup.-5 M                           2                   10.sup.-6 M                           5                   10.sup.-7 M                           5                   10.sup.-8 M                           6    ______________________________________

Morphological evaluation was made after another 3 days. As a result,whereas a tube meshwork was found all over in the untreated controlgroup, only independent local circles at most were observed in thetreatment groups, indicating inhibition of tube formation. The resultsare presented in Table 2. Referring to focal morphology (cellproliferation foci) in the table, the degree of intermeshing wasevaluated using the number of circles as an indicator. +++ stands forthe thorough integrity of a tube lining structure formed, ++ for theformation of circles, + for the formation of a lumen, and - forinhibition of tube formation.

                  TABLE 2    ______________________________________    Tube formation inhibitory action                         Focal morphology                         (cell proliferation    Treatment            focus)    ______________________________________    Untreated control group  +++    Test drug groups    Compound 1      10.sup.-4 M                             -                    10.sup.-5 M                             -                    10.sup.-6 M                             +                    10.sup.-7 M                             +    Compound 3      10.sup.-6 M                             -                    10.sup.-7 M                             +                    3 × 10.sup.-8 M                             +                    10.sup.-8 M                             +                    3 × 10.sup.-9 M                             ++    Untreated control group  +++    Test drug groups    Compound 2      10.sup.-6 M                             -                    10.sup.-7 M                             ++    Reference compound                    10.sup.-5 M                             +                    10.sup.-6 M                             ++                    10.sup.-7 M                             +++    ______________________________________

Compound 2 and 3 inhibited tube formation at 10⁻⁶ M and higherconcentrations and compound 1 did so at 10⁻⁵ M and higherconcentrations. The mechanism of neovascularization inhibition by thecompound of the invention remains to be fully elucidated as yet.

EXPERIMENTAL EXAMPLE 2 Effect on Body Weight Gain

Compound 1 was administered orally to 6-week-old male SD rats (5 animalsper group) daily and its effect on body weight gain was evaluated. As aresult, compound 1 did not affect body weight gain even at amultiple-dose level of 1000 mg/kg.

TEST EXAMPLE 3 Acute Toxicity (Mice)

The test compound was administered orally in a dose of 1 g/kg or 500mg/kg to 6-week-old male mice (BALB/c) in fasting state and theincidence of death was monitored for 1 week. The test compound wassuspended in 0.5% aqueous methylcellulose solution and administered in asingle dose of 500 or 1,000 mg/20 ml/kg. Compound 1 caused no death at 1g/kg.

Acute Toxicity (Rats)

Rats of either sex (SD strain, 280-360 g) were used in groups of 5. Theanimals were deprived of food from the previous day (16-18 hours beforethe experiment) and 1 g/kg of compound 1 was administered orally bygastric gavage. The animals were then monitored for death for 1 week. Asa result, no death was found at all.

The toxicity of the compound of the invention is, thus, very low.

BEST MODE OF CARRYING OUT THE INVENTION

The following production and working (formulation) examples are intendedto describe the present invention in further detail and should by nomeans be interpreted as defining the scope of the invention.

PRODUCTION EXAMPLE 1 2-Amino-4- N,N-bis(2-hydroxyethyl)amino!-6-(2,5-dichlorophenyl)-1,3,5-triazine (Compound 1)

To a mixture of 9.2 g of diethanolamine, 200 ml ofN,N-dimethylformamide, and 15 g of anhydrous potassium carbonate wasadded 20 g of 2-amino-4-chloro-6-(2,5-dichlorophenyl)-1,3,5-triazinewith stirring at room temperature and the mixture was further stirred atroom temperature for 7 hours. This reaction mixture was diluted with 2 L(liters) of water with stirring and then stirred for 1 hour. Theresulting crystals were collected by filtration, rinsed with water, anddried to obtain 24 g of white crystals. Those crystals wererecrystallized from methanol, collected by filtration, and dried to give21 g of the title compound as white crystals.

m.p. 199-200° C.

Elemental analysis (C₁₃ H₁₅ Cl₂ N₅ O₂)

Calcd. (%): C, 45.36; H, 4.39; N, 20.35

Found (%): C, 45.58; H, 4.33; N, 20.46

PRODUCTION EXAMPLE 22-Amino-4-(2,5-dichlorophenyl)-6-(3-hydroxymethyl-1-pyrrolidinyl)-1,3,5-triazinehydrochloride (Compound 2)

Except that 3-hydroxymethylpyrrolidine was used in lieu ofdiethanolamine, the reaction procedure of Production Example 1 wasotherwise repeated. The compound thus synthesized was dissolved inmethanol and while the resulting solution was cooled, 20% HCl/methanolwas added. The reaction mixture was then concentrated to about 1/10 ofthe initial volume and the resulting crystals were collected byfiltration to give the title compound as white crystals.

m.p. 241-243° C.

Elemental analysis (C₁₄ H₁₅ Cl₂ N₅ O.HCl)

Calcd. (%): C, 44.64; H, 4.28; N, 18.59

Found (%): C, 44.47; H, 4.34; N, 18.68

PRODUCTION EXAMPLE 3 2-Amino-4-(2,5-dichlorophenyl)-6-(2S,4R)-2-hydroxymethyl-4-hydroxy-1-pyrrolidinyl!-1,3,5-triazine(Compound 3)

Except that (2S,4R)-4-hydroxy-2-hydroxymethyl-pyrrolidine was used inlieu of diethanolamine, the procedure of Production Example 1 wasotherwise repeated to provide the title compound as white powders.

Elemental analysis (C₁₄ H₁₅ Cl₂ N₅ O₂. 1/2EtOH. 1/2H₂ O)

Calcd. (%): C, 46.40; H, 4.93; N, 18.04

Found (%): C, 46.36; H, 4.80; N, 18.24

H-NMR (CDCl₃) 67 : 1.7-2.0 (1H, m), 2.1-2.25 (1H, m), 2.67 (1H, bs),3.4-3.85 (3H, m), 3.95-4.25 (1H, m), 4.35-4.55 (2H, m), 5.53 (2H, d,J=11 Hz), 7.25-7.4 (2H, m), 7.65 (1H, d, J=19 Hz).

PRODUCTION EXAMPLE 4 2-Amino-4-(2,5-dichlorophenyl)-6-(2S,4R)-(2-hydroxymethyl-4-hydroxy-1-pyrrolidinyl!-1,3,5-triazine(Compound 3/Another Method)

Step 1 Using trans-4-hydroxy-L-proline methyl ester in lieu ofdiethanolamine, the procedure of Production Example 1 was otherwiserepeated to provide 2-amino-4-(2,5-dichlorophenyl)-6-(2S,4R)-2-methoxycarbonyl-4-hydroxy-1-pyrrolidinyl!-1,3,5-triazine aswhite powders.

Step 2 To a mixture of 20.9 g of lithium aluminum hydride and 1000 ml oftetrahydrofuran under ice-cooling and stirring, a solution of 100 g ofthe compound obtained in Step 1 in 300 ml of tetrahydrofuran was addedgradually dropwise at 0-5° C. and the reaction was carried out at thesame temperature for 3 hours. After the excess lithium aluminum hydridewas decomposed, the reaction mixture was filtered and the filtrate wasconcentrated. The residue was dissolved in ethyl acetate and thesolution was washed with water, dried, and concentrated to give a whitesolid. This solid was recrystallized from ethyl acetate, collected byfiltration, and dried to give 74.7 g of the title compound as whitecrystals.

m.p. 171-173° C.

Elemental analysis (C₁₄ H₁₅ Cl₂ N₅ O₂)

Calcd. (%): C, 47.21; H, 4.24; N, 19.66

Found (%): C, 47.19; H, 4.32; N, 19.55

α!²⁰ _(D) =-67.80° (MeOH, c=1.053)

FORMULATION EXAMPLE 1

Two (2) grams of compound 1 is weighed and mixed evenly with 70 g oflactose and 30 g of corn starch, followed by addition of 25 ml of 16%hydroxypropylcellulose solution. This mixture is agitation-granulated.After drying, the granules are size-selected, mixed with 2 g ofmagnesium stearate and 2 g of talc, and compressed with a rotary tabletmachine to provide tablets.

FORMULA

In 110 mg per tablet,

    ______________________________________    Compound 1        2 mg    Lactose           70 mg    Corn starch       30 mg    Hydroxypropylcellulose                      4 mg    Magnesium stearate                      2 mg    Talc              2 mg    ______________________________________

FORMULATION EXAMPLE 2

Four (4) milligrams of compound 1 is weighed and mixed evenly with 996mg of lactose to provide pharmaceutical powders.

INDUSTRIAL APPLICABILITY

Compared with the reference compound, the compound of the presentinvention has by far higher neovascularization inhibitory activity andis a safe compound with a low toxic potential as mentioned above.Therefore, the compound is useful for the treatment and prevention ofarteriosclerosis, tumors, Kaposi's sarcoma, diabetic retinopathy,rheumatoid arthritis, and other diseases in mammals inclusive of man.

What is claimed is:
 1. A method of effecting neovascular inhibition inhumans or animals which comprises the step of administering to saidhumans or animals a therapeutically effective amount of a compound ofthe following formula I! or a salt thereof, or a solvate thereof,##STR5## wherein R¹ represents hydrogen, optionally substituted alkyl,aralkyl, arylalkenyl, or aryl; R² represents optionally substitutedalkyl, aralkyl, arylalkenyl, or aryl; or R¹ and R² conjoinedly and takentogether with the adjacent N atom, i.e. in the form of NR¹ R², representa 4- through 8-membered cyclic amino group optionally containingnitrogen, oxygen, or sulfur as a ring member in addition to said N atomand optionally being further substituted.
 2. The method according toclaim 1, wherein R¹ represents (1) hydrogen, (2) alkyl which may besubstituted by a substituent selected from the group consisting ofhydroxy, alkoxy, amino, monoalkylamino, dialkylamino, arylamino, 4-through 8-membered cyclic amino, carboxy, carbamoyl, aryloxy, andacyloxy, (3) aralkyl, (4) arylalkenyl, or (5) aryl and R² represents (1)alkyl which may be substituted by a substituent selected from the groupconsisting of hydroxy, alkoxy, amino, monoalkylamino, dialkylamino,arylamino, 4- through 8-membered cyclic amino, carboxy, carbamoyl,aryloxy, and acyloxy, (2) aralkyl, (3) arylalkenyl, or (4) aryl.
 3. Themethod according to claim 1, wherein NR¹ R² represents a 4- through8-membered cyclic amino group which may have a substituent(s) selectedfrom the group consisting of hydroxy, oxo, carboxy, alkyl, hydroxyalkyl,aryloxyalkyl, aminoalkyl, alkylsulfonylaminoalkyl, alkylsulfonylamino,alkylsulfonylaralkyl, alkylsulfonyl, aryl, aralkyl, and 2-pyrimidinyl.4. The method according to claim 1, wherein R¹ and R² may be the same ordifferent and each represents hydroxyalkyl or NR¹ R² representspyrrolidin-1-yl, piperidino, or morpholino, which may be substituted. 5.The method according to claim 1, wherein NR¹ R² representspyrrolidin-1-yl which may be substituted by hydroxy, hydroxyalkyl, oxo,alkyl, amino, or aminoalkyl.
 6. The method according to claim 1, whichcomprises administering a compound selected from the group consisting of2-amino-4- N, N-bis (2-hyroxyethyl)amino!-6-(2,5-dichlorophenyl)-1,3,5-triazine,2-amino-4-(2,5-dichlorophenyl)-6-(3-hydroxymethyl-1-pyrrolidinyl)-1,3,5-triazinehydrochloride, and 2-amino-4-(2,5-dichlorophenyl)-6-(2S,4R)-2-hydroxymethyl-4-hyroxy-1-pyrrolidinyl!-1,3,5-triazine.
 7. Themethod according to claim 1, which comprises administering2-amino-4-(2,5-dichlorophenyl)-6- (2S,4R)-2-hydroxymethyl-4-hydroxyl-1-pyrrolidinyl!-1,3,5-triazine.
 8. Amethod of treating arteriosclerosis in humans or animals whichcomprising administering to said humans or animals a therapeuticallyeffective amount of a compound of the following formula (I) or a saltthereof, or a solvate thereof: ##STR6## wherein R¹ represents hydrogen,optionally substituted alkyl, aralkyl, arylalkenyl, or aryl; R²represents optionally substituted alkyl, aralkyl, arylalkenyl, or aryl;or R¹ and R² conjoinedly and taken together with the adjacent N atom,i.e. in the form of NR¹ R², represent a 4- through 8-membered cyclicamino group optionally containing nitrogen, oxygen, or sulfur as a ringmember in addition to said N atom and optionally being furthersubstituted.
 9. A method of treating Kaposi's sarcoma in humans oranimals which comprising administering to said humans or animals atherapeutically effective amount of the compound of the followingformula (I) or a salt thereof, or a solvate thereof ##STR7## wherein R¹represents hydrogen, optionally substituted alkyl, aralkyl, arylalkenyl,or aryl; R² represents optionally substituted alkyl, aralkyl,arylalkenyl, or aryl; or R¹ and R² conjoinedly and taken together withthe adjacent N atom, i.e. in the form of NR¹ R², represent a 4- through8-membered cyclic amino group optionally containing nitrogen, oxygen, orsulfur as a ring member in addition to said N atom and optionally beingfurther substituted.
 10. A method of treating diabetic retinopathy inhumans or animals which comprising administering to said humans oranimals a therapeutically effective amount of a compound of thefollowing formula (I) or a salt thereof, or a solvate thereof ##STR8##wherein R¹ represents hydrogen, optionally substituted alkyl, aralkyl,arylalkenyl, or aryl; R² represents optionally substituted alkyl,aralkyl, arylalkenyl, or aryl; or R¹ and R² conjoinedly and takentogether with the adjacent N atom, i.e. in the form of NR¹ R², representa 4- through 8-membered cyclic amino group optionally containingnitrogen, oxygen, or sulfur as a ring member in addition to said N atomand optionally being further substituted.
 11. A method of treatingrheumatoid arthritis in humans or animals which comprising administeringto said humans or animals a therapeutically effective amount of acompound of the following formula (I) or a salt thereof, or a solvatethereof, ##STR9## wherein R¹ represents hydrogen, optionally substitutedalkyl, aralkyl, arylalkenyl, or aryl; R² represents optionallysubstituted alkyl, aralkyl, arylalkenyl, or aryl; or R¹ and R²conjoinedly and taken together with the adjacent N atom, i.e. in theform of NR¹ R², represent a 4- through 8-membered cyclic amino groupoptionally containing nitrogen, oxygen, or sulfur as a ring member inaddition to said N atom and optionally being further substituted. 12.The method according to claim 1, comprising administering apharmaceutical composition comprising a compound of the formula (I) or asalt thereof, or a solvate thereof and a pharmaceutically acceptablenontoxic carrier or vehicle.
 13. The method according to claim 12,wherein the administration is oral, parenteral or rectal.
 14. The methodaccording to claim 8, comprising administering a pharmaceuticalcomposition comprising a compound of the formula (I) or a salt thereof,or a solvate thereof and a pharmaceutically acceptable nontoxic carrieror vehicle.
 15. The method according to claim 14 wherein theadministration is oral, parenteral or rectal.
 16. The method accordingto claim 9, comprising administering a pharmaceutical compositioncomprising a compound of the formula (I) or a salt thereof, or a solvatethereof and a pharmaceutically acceptable nontoxic carrier or vehicle.17. The method according to claim 16, wherein the administration isoral, parenteral or rectal.
 18. The method according to claim 10,comprising administering a pharmaceutical composition comprising acompound of the formula (I) or a salt thereof, or a solvate thereof anda pharmaceutically acceptable nontoxic carrier or vehicle.
 19. Themethod according to claim 18, wherein the administration is oral,parenteral or rectal.
 20. The method according to claim 11, comprisingadministering a pharmaceutical composition comprising a compound of theformula (I) or a salt thereof, or a solvate thereof and apharmaceutically acceptable nontoxic carrier or vehicle.
 21. The methodaccording to claim 20, wherein the administration is oral, parenteral orrectal.